Melatonin and aggressive behavior: A systematic review of the literature on preclinical and clinical evidence

Abstract The melatonin system and circadian disruption have well‐established links with aggressive behaviors; however, the biological underpinnings have not been thoroughly investigated. Here, we aimed at examining the current knowledge regarding the neurobiological and psychopharmacological involvement of the melatonin system in aggressive/violent behaviors. To this end, we performed a systematic review on Embase and Pubmed/MEDLINE of preclinical and clinical evidence linking the melatonin system, melatonin, and melatoninergic drugs with aggressive/violent behaviors. Two blinded raters performed an independent screening of the relevant literature. Overall, this review included 38 papers distributed between clinical and preclinical models. Eleven papers specifically addressed the existing evidence in rodent models, five in fish models, and 21 in humans. The data indicate that depending on the species, model, and timing of administration, melatonin may exert a complex influence on aggressive/violent behaviors. Particularly, the apparent contrasting findings on the link between the melatonin system and aggression/violence (with either increased, no, or decreased effect) shown in preclinical models underscore the need for further research to develop more accurate and fruitful translational models. Likewise, the significant heterogeneity found in the results of clinical studies does not allow yet to draw any firm conclusion on the efficacy of melatonin or melatonergic drugs on aggressive/violent behaviors. However, findings in children and in traits associated with aggressive/violent behavior, including irritability and anger, are emerging and deserve empirical attention given the low toxicity of melatonin and melatonergic drugs.


| INTRODUCTION
Melatonin (N-acetyl-5-methoxytryptamine) (MLT) is a neurohormone synthesized in the pineal gland from its precursor serotonin (5-HT) primarily during the dark phase of the light/dark cycle. In addition, extrapineal synthesis of MLT has been reported in the retina, Harderian gland, and gastrointestinal tract. 1 MLT regulates several neurophysiological functions, including sleep, mood, circadian rhythm, pain, as well as reproduction, and possesses anti-inflammatory and antioxidant properties. [2][3][4][5][6][7] MLT functions are mainly mediated by two seven-transmembrane G-protein coupled receptors named MT 1 and MT 2, which differ in terms of molecular structure, chromosomal localization, interactions with different intracellular signaling proteins, and pharmacological affinity to MLT. A detailed description of the differences between these two MLT receptor subtypes lies outside the scope of this systematic review and can be found elsewhere. 3,8 The presence of MLT receptors in different brain regions as well as in peripheral tissues and organs may account for the different pathophysiological effects of MLT in the human body. 9 In humans, the onset of MLT secretion is around 21:00-22:00 h, it reaches a peak level of 80-120 pg/ml in the blood between 24:00 and 3:00 h, and the offset of its secretion is in the morning at 7:00-9:00 h, when its serum levels fall down to 10-20 pg/ml. 10 This circadian production of MLT is under the control of the suprachiasmatic nuclei (SCN), the master clock, in the hypothalamus. The SCN receives input from the retinohypothalamic tract to synchronize with the external light/dark cycle and is connected with the pineal gland through a multisynaptic pathway involving the paraventricular nucleus of the hypothalamus, the sympathetic preganglionic neurons of the intermediolateral cell column in the spinal cord, and the sympathetic noradrenergic neurons of the superior cervical ganglion (SCG). 11 Once synthesized, MLT has a feedback control on the activity of the SCN by acting on its two receptors, both located at the level of the nucleus. 12 Growing evidence indicates a strict link between circadian rhythms, behavior, emotions, and mental health/illness. 13 Consequently, a disruption of the physiological circadian rhythms seems to be implicated in the pathophysiology of psychiatric disorders such as anxiety disorders, major depressive disorder, and bipolar disorder. 14,15 For instance, there is consistent evidence that the evening chronotype is associated with the severity of psychiatric disorders and may represent a risk factor for their development. 16 Of note, it has been demonstrated that aggressive behavior tends to be more pronounced in children and adolescents with higher eveningness, for example, those that stay up late at night, and are more mentally and physically active in the late afternoon or evening. 17 This is of great relevance as aggression is a behavioral disturbance shared by several psychiatric disorders. Despite the well-established clinical link between circadian disruption and aggression, 18 a limited number of studies investigated its biological underpinnings (for a review, see Bronsard & Bartolomei 19 ). In light of the role of MLT as a zeitgeber for the circadian system, here, we aimed to review current knowledge of the involvement of the MLT system in aggressive/violent behavior by looking at the correlations between changes in MLT levels or MLT receptors and the development of aggressive/violent behavior, and at the possible effects of MLT or MLT receptors ligands in the treatment of aggressive individuals. Importantly, we used a translational approach integrating animal and human findings allowing us to gain novel insights on the role of MLT in the neurobiology and psychopharmacology of aggressive behavior.

| Aggressive behavior
The need of increasing the comprehension of the neurobiological underpinnings of aggressive behavior stems from the presence of several unmet goals in the treatment, prevention, and the consequent reduction of the associated socioeconomic burden of this devastating behavioral disturbance. This delay has been determined by several factors. Primarily, the substantial heterogeneity in the phenotypic definition of aggressive behavior decreases the power of studies exploring its biological underpinnings. 20 This derives mainly, but not exclusively, from the lack of a consensus on the clinical definition (criteria) of aggressive behavior between the different disciplines examining this phenomenon. 21,22 Indeed, only recently the harmonization of diverse types of measures of aggression has allowed estimating the impact of key environmental components such as the sibling interaction effect. 23 In addition, the phenotypic heterogeneity has significantly hindered the identification of reliable biomarkers of aggression. 24 Although multi-omics approaches are starting to produce promising results, 25,26 accurate predictive models are still lacking. This is in part a consequence of the complex interaction between genetic susceptibility and environmental factors that determines the biological make-up of aggression. 27 The multifaceted neurobiology of aggression involves multiple neurochemical pathways, with certain forms, such as impulsive aggression, possibly associated with dysregulation of the serotonergic signaling system. 22 However, even for those pathophysiological mechanisms that have been elucidated, there is a poor characterization of causality as well as of the molecular mechanisms leading to treatment efficacy. 22 Of note, little is known on the role of the MLT system in aggression. Given that phylogenetically MLT is an ancient molecule 28 and that aggression itself is a primeval ubiquitarian behavioral trait in human and nonhuman vertebrates, 29 it is conceivable that converging data from clinical and preclinical studies can better inform our knowledge of the biological architecture of aggression. In this context, here, we performed a systematic review of clinical studies and animal models exploring: (1) alterations of the MLT system in aggression and (2) testing whether the perturbation of the MLT system with exogenous MLT administration or MLT receptors ligands can attenuate (or exacerbate) aggression/violence.

| Search methodology
We performed a systematic review in Pubmed/MEDLINE and Embase according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) (Figure 1) using the following search terms: "melatonin," "MT 1 receptor," and "MT 2 receptor" along with "aggression," "violence," "impulsivity," "agitation," "anger," and "irritability." This search strategy was augmented by identifying additional studies replacing the term "melatonin" with "agomelatine," "ramelteon," or "tasimelteon" that are all nonselective MT 1 and MT 2 receptors agonists approved for clinical use in humans, 30 as well as with "luzindole" or "4-phenyl-2-propionamidotetraline (4P-PDOT)" that are a nonselective MT 1 and MT 2 receptors antagonist and a selective MT 2 receptor antagonist, respectively. Further, we examined the references listed in the included papers to identify studies meeting our inclusion and exclusion criteria.
The search was last performed in August 2021, specifically focusing on records regarding trials, case reports, and studies on clinical and preclinical models. Systematic reviews and meta-analysis were discarded. We first screened titles, and those clearly not in line with the purpose of the review were excluded. Then abstracts were assessed, and lastly, full texts were read, eventually leading to the inclusion or exclusion of the papers, according to the criteria established before the online search. A systematic review of the literature was performed on Medline and EMBASE. The screening of the literature was performed in blind by two investigators (P.P. and S.C.). In the case of disagreement, a third reviewer (M.M.) assessed the paper and a consensus was reached.

| Inclusion and exclusion criteria
To be included in the review, research studies had to either: (a) investigate the role of the melatonergic system in aggression in clinical studies or animal models or (b) test the effect of MLT or pharmacological modulators of the MLT receptors and (c) be written in English. We excluded reviews and meta-analyses of the literature.

| Data extraction
The following data considered relevant for the systematic search were extracted from each study and tabulated in a data management software: study, animal species, type of test, the dose of MLT (or treatment), outcome.

| Assessment of risk of bias
The risk of bias assessment in randomized controlled trials was performed using the RoB v.2 tool. 31 Bias domains included in this tool are randomization, deviation from intended interventions, missing data, measurement of outcomes, and selection of reported results. For each tested domain, the relative risk of bias is assigned on a three-level scale ranging from "Low" to "High" risk of bias. When no information is available to judge a particular item, a "No information" flag is used instead. For each paper, an "overall risk" is also attributed. The risk of bias for nonrandomized studies was evaluated with the ROBINS-I assessment tool, 32 an instrument reporting on bias arising from confoundings, selection of study participants, classification of interventions, deviation from intended interventions, missing data, measurement of outcomes, and selection of reported measures. For each tested domain is assigned a score on a four-level scale ranging from "Low" to "Critical" risk of bias. As previously described for the RoB v. 2, the overall risk of bias is also attributed, and in case of missing data, a "No information" flag is available. A "traffic plot" summary for each test was developed with the web-based robvis tool. 33

| RESULTS
Our systematic review identified a total of 38 papers distributed between clinical and preclinical models ( Figure 1) showing an association between changes within the MLT system and aggression or an effect of PARIBELLO ET AL. either MLT or compounds targeting MLT receptors on aggression. Eleven papers specifically addressed the existing evidence in murine models, five on fish models, and 21 on humans. We present the main results of these studies in the following sections.

| MLT and aggression: Preclinical findings
Preclinical studies examining the effects of MLT administration or changes within the MLT system on F I G U R E 1 PRISMA 2020 flow diagram for the systematic review aggressive behavior have been conducted in rodents mostly using the resident-intruder paradigm (Table 1), but interestingly, many studies have also been performed in different species of fish, including the cichlid fish and the rainbow trout (Table 2).

| MLT and aggression: Preclinical findings in rodents
We identified 11 studies testing the effects of MLT on aggressive behavior in rodents. Demas et al. 34 studied in the resident-intruder paradigm the effect of a subcutaneous injection of 15 μg MLT for 10 days, reflecting typical short-day patterns in Siberian adult male Hamsters housed on long-day photoperiod. They detected an MLT-induced decrease in the latency to attack and an increase in the number of attacks to the intruder. Notably, the pro-aggressive effects of exogenous MLT were attenuated by bilateral adrenalectomies but not by bilateral adrenal demedullations, indicating the involvement of adrenocortical rather than adrenomedullary hormones. 34 Furthermore, the effect of photoperiod was studied in female Golden Hamsters showing that shortday animals had a higher ratio of offensive to defensive behaviors than long-day animals. 35 Interestingly, this difference was blunted in pinealectomized animals and restored by exogenous injection of MLT, thus demonstrating a pro-aggressive role for MLT. 35 These effects were independent of gonadal effects as further confirmed by experiments conducted in ovariectomized hamsters. 35 Similar findings have also been demonstrated in shortday male Syrian hamsters, which displayed higher levels of aggressive behavior than long-day animals, and the injection of MLT in long-day hamsters significantly increased aggression. 36 Importantly, these differences in aggressive behavior were not correlated to changes in the levels of testosterone but likely to changes in the HPA axis leading to altered production of adrenal hormones, in particular glucocorticoids. 36 An interesting experiment by Heinzeller et al. 37 examined the effects of single and repeated aggressive encounters on the synthesis of MLT in the pineal gland. The authors found an elevation of MLT immediately at the end of the encounter, followed by a decline in the levels of the neurohormone. Conversely, animals with superior cervical ganglionectomy, and thus sympathetic denervated pineal gland, had a fall in MLT levels at the end of the encounter. 37 Repeated aggressive encounters produced a 2-h delay in the nocturnal rise of pineal MLT levels. 37 MLT also increased aggressive behavior in California mice housed under long-day photoperiod, but interestingly, this pro-aggressive effect was only partially blocked by the nonselective MT 1 -MT 2 receptors antagonist luzindole, which per se at the employed dose (40 mg/kg) did not affect aggressive behavior. 38 In addition, the authors found that these behavioral effects of MLT were not mediated by suppression of estrogen-dependent genes that were previously associated with increased aggression. 38 Increased impulsivity is one of the risk factors for aggressive and violent behaviors. 39,40 Loiseau et al. 41 found that MLT and agomelatine, a nonselective MT 1 and MT 2 receptors agonist and an antagonist to 5-HT 2B/2C receptors, reduced impulsive-related behavior of Wistar AF rats measured as an increased number of choices of the large-but-delayed reward in a T-Maze apparatus. Interestingly, the effects of MLT on the number of choices were not blocked by the MT 1 and MT 2 antagonist S22153, suggesting that the observed effects of MLT on impulsive behavior are not MLT receptors mediated. Paterson et al. 42 investigated in mice the involvement of the pineal-adrenal axis in relation to territorial aggression. They were able to confirm the enhancing-fighting properties of MLT in rodents, as well as to demonstrate that these behavioral effects rely on the inhibitory action of this neurohormone on the adrenal cortex. 42 Indeed, no effects of MLT on aggression were observed in animals with adrenalectomy or treated with aminoglutethimide, a steroid synthesis inhibitor that depletes adrenocortical hormones. 42 In addition, no effects of MLT were seen in animals undergoing repeated restraint stress and sham-operated animals for ADX. 42 Rendon et al. 43 investigated the link between sex steroids, MLT, and aggressive behavior in female Siberian hamsters, a highly territorial species. They found that shortday females were more aggressive than long-day females, and this elevated aggression was concomitant with a dehydroepiandrosterone (DHEA)-specific increase in adrenal responsiveness. In addition, injection of MLT in long-day females significantly increased aggressive behavior and circulating levels of DHEA. 43 Interestingly, the elevation of circulating DHEA induced by MLT is due to increased adrenal and reduced gonadal DHEA release in short-day females and the opposite in long-day females. The effect of MLT on agonistic behavior and social dominance and its possible interaction with testosterone was studied in longtailed hamsters. 44 In both short-and long-day photoperiods, intact males displayed more agonistic behavior than castrated males, but no differences were observed between pairs of long-and short-day intact or castrated animals. 44 Within castrated animals, short-day males showed a higher win rate against long-day animals. 44 Six weeks of MLT implantation increased aggressive and dominant behaviors more in long-than in short-day castrated male hamsters, whereas the implantation of testosterone into short-day  45 proposed MLT as a possible modulator in the metabolism and synthesis of androgens associated with an aggressive encounter. MLT also appears to be able of reducing methamphetamine-induced aggression in the resident-intruder paradigm. 46 Under the same experimental conditions, the hippocampal levels of dopamine, serotonin, and some of their metabolite levels were influenced by MLT exposure.

| MLT and aggression: Preclinical findings in fish
Larson et al. 47 investigated in rainbow trouts, a species displaying strong dominance hierarchy, whether circulating levels of MLT were associated with the dominancesubordination relationship. Subordinate fishes displayed increased night-time levels of MLT, and in both dominant and subordinate fishes, a positive correlation between MLT and cortisol plasma levels was observed during the day but not during the night. 47 Audira et al. 48 reported a 12-fold reduction in MLT levels in Zebrafish leptin knockout mutant. Circadian rhythm appeared significantly altered as compared with controls, with aggressive behaviors also appearing altered: considering the setting and the possible confoundings, more data might be required to better establish a clearer causal link. MLT exposure appeared associated with a reduction in aggressiveness and lipid peroxidation in Brycon amazonicus, a species known for engaging in cannibalistic behaviors and for being subject to oxidative stress related to being kept in artificial environments. 49 As feeding fish with a tryptophan-enriched diet was known to decrease aggressive behavior, Lepage et al. 50 investigated whether this effect was mediated by the central serotonergic and/ or MLT systems of which tryptophan is the common precursor. Tryptophan-enriched diet produced a suppression of aggressive behavior and an increase of plasma MLT levels. Conversely, the exogenous administration of MLT producing a similar elevation of plasma MLT levels was ineffective in suppressing aggressive behavior. Citalopram, a serotonin re-uptake inhibitor, instead induced an aggression-suppressive effect similar to that produced by the tryptophan-enriched diet, indicating that the effects of the amino acid were likely mediated by serotonin and not by MLT. 50 Munro 51 instead showed in another fish, the Aequidens pulcher, that MLT reduced aggression similar to 5-HT, and that the anti-aggressive effects of 5-HT were likely mediated by its conversion into MLT. Indeed, simultaneous treatment with 5-HT and S-adenosyl homocysteine, an inhibitor of the conversion of 5-HT into MLT, blocked the inhibitory effects of 5-HT on aggression. 51

| MLT and aggression: Clinical findings
MLT and melatonergic agonists have been evaluated in diverse clinical settings as summarized in Table 3. Although case-report studies might be of limited scientific validity and may lead to over-interpretation of the findings, we decided to include them as this is the first systematic work examining the link between MLT and aggression. Thus, it may be used to generate new hypotheses for future studies. Asano et al. 52,53 reported two case reports suggesting the possible efficacy of ramelteon in the management of aggressivity. In the first one, ramelteon significantly improved agitation/aggression and irritability in a 79-year-old man patient affected by Alzheimer's disease (AD) with behavioral and psychological symptoms of dementia. The second case report described an improvement in irritable and violent behaviors with ramelteon in a 16-year-old boy with high-functioning autistic disorder diagnosed at the age of 5 years. Parallel to this decrease in impulsive/aggressive behaviors, in both cases, an improvement of sleep was observed (i.e., a reduction of the latency to fall asleep and an increase of the sleep duration). An open-label study involving individuals affected by Smith-Magenis syndrome found that tasimelteon resulted in an improvement in parentrated behavior and sleep quality as compared to baseline. 54 In contrast, in a double-blind randomized placebocontrolled study of 41 patients with severe AD, MLT did not induce any improvement in neither sleep nor agitation. 55 Several papers reported on MLT's impact on the risk of postoperative delirium. In a randomized controlled trial involving 148 children (36-37 children per tested dose of MLT) undergoing anesthesia, premedication with MLT reduced in a dose-dependent fashion the incidence of delirium. 56 Similar findings were also obtained by Ozcengiz et al. 57 in a placebo-controlled, double-blinded trial in 100 children and by Samarkandi et al., 58 who found that premedication with MLT in children undergoing general anesthesia for minor elective surgery reduced or prevented postoperative excitement. Of note, this reduced postoperative excitement was greater with MLT also compared to the benzodiazepine midazolam, indicating that the effect of MLT was not subsequent to increased sedation of the children. In a more recent work, MLT reduced the incidence of sevoflurane-induced agitation among 120 children (30 children per group) undergoing adenotonsillectomy. 59 On the contrary, Jaiswal et al. did not find any effect for ramelteon in the prevention of postoperative delirium among individuals undergoing elective pulmonary thromboendoarterectomy. 60 Similarly, no effect was reported for ramelteon in the incidence of postoperative delirium in a further randomized, placebo-controlled trial among pediatric individuals undergoing tonsillectomy. 61 A retrospective, observational study investigated the preventive effect of MLT, ramelteon, or placebo in the incidence of delirium among individuals admitted to a single-center intensive care unit (ICU Aggressive behavior is also one of the most prominent symptoms in children with autism. 65 An open-label study aimed at testing the possible therapeutic effects of MLT on sleep and behavioral symptoms in children with autism. 66 MLT treatment (1-6 mg) appeared associated with a tendency (p = .073) to reduce aggressive behaviors, but with no noticeable effect on self-injurious behaviors. A further open-label study, 67      discontinuation. Similarly, the results presented by Schroder et al. 68 suggest that prolonged-release MLT minitablets at doses of 2-5 mg, may decrease aggression among children affected by an autism spectrum disorder or by Smith-Magenis syndrome. A randomized, double-blind, placebo-controlled trial involving male volunteers, reported a higher propensity for choosing to administer a high punishment to an opponent in the Taylor Aggression Paradigm with MLT exposure. 69 In a randomized, placebo-controlled trial, van der Heijden et al. 70 evaluated the efficacy of 3-6 mg MLT in influencing sleep, behavior, quality of life, and cognitive performances among children affected by attention-deficit/ hyperactivity disorder (ADHD). The intervention improved sleep parameters, but no effect was observed in the remaining outcomes. 70 Other studies tested whether nonselective MLT receptors ligands can reduce the severity of aggression. In an openlabel trial involving 10 children with ADHD, agomelatine appeared associated with reduced fidgeting and only slight sedative effects, suggesting that the observed decrease in irritability/agitation may be independent of the possible sedative effects of the drug. 71 A case report described a reduction in challenging behaviors using agomelatine in a 61-year-old patient with severe brain damage due to a subarachnoid hemorrhage. 72 Importantly, this effect was maintained during the entire 18 months follow-up. Finally, a retrospective study of 125 patients with delirium tested whether ramelteon could decrease the need for an antipsychotic prescription for agitation. 73 The authors found that ramelteon appeared associated with a lower incidence of antipsychotic use compared to controls.

| Risk of bias of the included studies
The risk of bias for all the included randomized clinical trials was judged to be low, with one trial not specifying the employed randomization strategy. 69 The results are summarized in Figure 2. A total of eight nonrandomized studies were eligible for the assessment according to the ROBINS-I tool. One before-after study was judged at low risk of bias. 66 The remaining three before-after trials were judged to be at serious risk of bias. One of them presented an inadequate assessment at baseline (i.e., a singular evaluation and no follow-up time at baseline) and a high risk of measurement bias (i.e., unblinded raters evaluating subjective outcome scales). 67 The remaining two presented a serious risk for possible measurement bias (i.e., subjective outcome measures evaluated by unblinded raters). 54,74 Three studies were judged at moderate risk for confounding by indication and for the assignment of intervention status due to the retrospective design. 62,64,73 An open-label, placebocontrolled study was at serious risk of confoundings and measurement bias as no information was available on the total number of potentially eligible individuals. 71 The results are summarized in Figure 3.

| DISCUSSION
Our systematic review highlighted a series of important and novel findings. First, while a link between the photoperiod and thus variation in the physiological levels of MLT, and levels of aggressive behavior has been demonstrated in F I G U R E 2 Illustration of the risk of bias for randomized clinical trials several studies in different species, there is still inadequate and limited evidence to draw any firm conclusion about a possible anti-aggressive effect of MLT or MLT receptors agonists such as ramelteon or agomelatine. Most of the effects of MLT are mediated by its two receptors, but their possible selective role in aggressive/violent behaviors has not been explored yet. Growing evidence suggests that the two MLT receptors may control/modulate different physiopathological processes at central and peripheral levels. 4,[75][76][77][78][79][80][81][82] Importantly, selective MLT receptors agonists/ partial agonists seem to be therapeutically superior to the nonselective MLT. For example, MT 2 receptor partial agonists showed more potent hypnotic 77,83 and analgesic 80,81 effects than MLT. Given the link between the MLT system and aggressive behavior, it will be critical to understand if both MT 1 and MT 2 receptors or only one of the two subtypes are implicated, especially considering the possible development of new treatments for aggressive behaviors. Thanks to the recent advancement in medicinal chemistry and MLT ligands, we now have the availability of selective MT 1 and MT 2 agonists as well as antagonists. Thus, it will be possible to investigate the two MLT receptor subtypes' effects on aggressive behavior. In addition, research on aggressive behavior in MLT receptors knockout mice 84,85 as well as using selective or nonselective MT 1 and MT 2 antagonists could provide further insights. To date, we found only one preclinical study in California mice, which tested the nonselective MT 1 and MT 2 receptors antagonist luzindole finding no effect of the drug on aggressive behavior. 38 However, only one single and likely very high dose (40 mg/kg) was tested. 38 MLT receptors have been localized in brain regions 9 intimately implicated in aggression including the prefrontal and frontal cortices, the hippocampus, the hypothalamus, and the raphe nuclei. 40,86 They modulate differently monoaminergic 75,87,88 and glutamatergic 81 neurotransmissions. Consequently, it is plausible to expect that MT 1 and MT 2 receptors may have a role in the pathophysiology of aggressive behavior.
Serotonin (5-HT) is the most widely studied neurotransmitter in aggression. 22,24 It should be emphasized that 5-HT is the precursor of MLT and thus variations in the levels of 5-HT are likely to influence the levels of MLT. Although many studies have correlated cerebrospinal fluid levels of 5-HT or its metabolites to aggression, no research has investigated possible variations in the levels of MLT. In addition, following a tryptophan depletion which lowers central 5-HT levels, an increase in aggression has been observed. 89,90 But a tryptophan depletion also brings to a decrease in nocturnal MLT secretion. 91 Therefore, it would be interesting also to differentiate the possible effects of MLT from that of 5-HT in the increased aggression following the tryptophan depletion.
Preclinical studies in different rodents but also in fish have produced contrasting findings showing either increased, decreased, or no effect of MLT on aggression. We think that this apparently conflicting evidence may in part derive from the different experimental conditions employed, which may have strongly impacted the outcomes, as well as on the rodent model (nocturnal vs. diurnal). For example, studies differ significantly in the tested doses of MLT, the timing of the day of MLT administration, the time of the day, and the duration of the light/dark cycle in which the animals are tested. Sexspecific effects are also worth further investigation for their possible clinical implications. Furthermore, several F I G U R E 3 Illustration of the risk of bias for nonrandomized studies strains of mice produce no or low amounts of MLT due to defects in the activity of one or both the enzymes that sequentially transform 5-HT into N-acetylserotonin (the enzyme serotonin N-acetyltransferase) and then Nacetylserotonin into MLT (the enzyme hydroxyindole Omethyltransferase). Mice of the C57BL/6 strain that have been largely used in the preclinical studies here reviewed are indeed among the MLT-deficient inbred strains. 92 As very few studies investigated the circulating levels of MLT, none examined the MLT concentrations in the brain at the time of testing, and the circadian variability in the levels of MLT has a strong implication on behavior and many other physiological functions (i.e., the expression of MLT receptors), the assessment of MLT levels in future studies may help further clarify these discrepancies. Finally, studies on the possible effects of MLT on aggression in nonmammalian vertebrates such as birds, reptiles, and amphibians are currently missing and need to be conducted to increase our understanding of the link between the MLT system and aggressive behavior.
In the literature here reviewed, we found discrepancies not only among preclinical studies but also between preclinical and clinical studies. For instance, while clinical studies highlighted a possible effect of MLT in reducing aggressive behavior, several preclinical studies in rodents reported the opposite. In this context, it is plausible that most of the observed discrepancies may rely on the difference in the circadian systems of these species (i.e., nocturnal vs. diurnal), rather than in the methodology. Indeed, virtually all rodents are nocturnal, meaning that high levels of MLT occur at times in which there is a peek in their behavioral activity, which includes also interactions with conspecifics and thus likely aggression. In contrast, in humans (Table 3) as well as in some of the fish studies (Table 2) here presented, the circadian system is primarily diurnal, meaning that the circulating levels of MLT are the highest when these animals are the least active. Moreover, given the link among MLT, circadian system, and sleep, and the fact that sleep dysfunctions can be a risk factor for aggressive behavior, 93,94 it can be hypothesized that the putative mechanism of MLT on aggressive behavior in humans relates to the quality of sleep, whereas in rodent models it is more likely related to serving a function during times of the day (or seasons) when heightened aggression is selected for. Future studies are warranted to examine this hypothesis as an example by looking at the link between the MLT system and aggression in diurnal animals (e.g., Meriones unguiculatus, Octodon degus, Arvicanthis niloticus, and Ammospermophilus leucurus) 95 to see whether in these animals the findings more closely match with those found in human studies.
Among the clinical studies included in Table 3, those conducted in children/adolescents affected by different psychiatric disorders in which aggressive traits may be present, including oppositional defiant disorder, ADHD, conduct disorder, and autistic spectrum disorders, have shown a global agreement in highlighting positive effects of MLT or melatonergic drugs in controlling aggressive/ violent behavior or traits associated with aggression such as irritability, anger, and agitation. Future randomized double-blind placebo trials are thus welcome to support these encouraging findings especially because the prevalence of these developmental disorders is increasing worldwide, and up to now, there are no yet approved drugs to control aggressive-like traits in children/adolescents. Of note, it should be emphasized that long-term treatment with MLT seems to be generally well-tolerated and safe. 30,96,97 Studies also conducted in the elderly have suggested positive effects of MLT or melatonergic compounds alone or in combination with other drugs in controlling aggression, irritability, or agitation associated with different medical conditions. Several lines of evidence also suggest the efficacy of MLT use and its analog in preventing postoperative delirium, and these findings should also be further explored. 98 All the included randomized controlled trials appear to be at low risk of bias, and therefore, despite the small sample sizes involved, the resulting evidence is deemed promising. However, evidence is still preliminary and more controlled trials are needed. The quality of the included nonrandomized trials is more heterogeneous, and thus the interpretation of their results appears more challenging.
In conclusion, although there are still many unanswered questions, this review reports current evidence on the neurobiological and psychopharmacology involvement of MLT and its receptors in aggressive/violent behavior, and given the encouraging findings, highlights the importance of conducting more studies in this still unexplored field of research.

ACKNOWLEDGMENT
The authors wish to thank Dr. Greta Forcaia for her assistance in preparing the tables and the initial literature search. Open access funding provided by Universita degli

CONFLICTS OF INTEREST
The authors have no conflicts of interest to declare.

AUTHOR CONTRIBUTIONS
Pasquale Paribello performed the literature search, contributed to the assessment of available evidence, and prepared the first draft of the manuscript. Mirko Manchia contributed to the literature search and the assessment of the available evidence and prepared the PARIBELLO ET AL.

| 17 of 21
Studi di Padova within the CRUI-CARE Agreement.
first draft of the manuscript. Marta Bosia, Federica Pinna, and Bernardo Carpiniello contributed to data collection and interpretation and critically revised the manuscript. Stefano Comai designed and oversaw the study, contributed to the literature search and the assessment of the available evidence, and prepared the first draft of the manuscript. All authors reviewed and approved the final version of this manuscript.

DATA AVAILABILITY STATEMENT
Data sharing is not applicable to this article as no data sets were generated or analyzed during the current study.